Compounds, compositions and methods for treating influenza

ABSTRACT

A compound produced by the process comprising the step of reacting a starting compound of the formula:                    
     with FeCl 3  in the presence of acetic acid, wherein the reaction mixture is heated and the temperature of the reaction mixture is about 85° C.to about reflux temperature, R 1  and R 2  in the above formula being as defined in the present specification.

The present application is a divisional of U.S. patent application Ser.No. 09/371,057, filed Aug. 9, 1999, now U.S. Pat. No. 6,180,628 which isa continuation of U.S. patent application Ser. No. 09/082,656, filed May21, 1998, now U.S. Pat. No. 5,935,957, which is a continuation of U.S.patent application Ser. No. 08/858,649, filed May 19, 1997, now U.S.Pat. No. 5,821,243, which is a continuation-in-part of U.S. applicationSer. No. 08/681,289, filed Jul. 22, 1996 (now abandoned).

FIELD OF THE INVENTION

The present invention relates to compounds, compositions and methods forthe treatment of influenza infection. In particular, the presentinvention relates to novel pyridazine derivatives, pharmaceuticalcompositions containing such derivatives and their use in treatinginfluenza infection and other viral diseases.

BACKGROUND OF THE INVENTION

There are three known influenza-type viruses which affect human beings:Influenza A, B and C. Influenza A viruses have been isolated from manyanimal species in addition to humans, while the influenza B and Cviruses infect mainly humans. The influenza viruses are envelopedviruses containing negative single-stranded RNA's which are segmentedand encapsidated. The influenza virus envelope is characterized by thepresence of two surface glycoproteins: hemagglutinin and neuraminidase.The influenza A and B virions are pleomorphic and are usually 80-120 nmin diameter. The influenza C virion has many distinctive properties andis thus distinguished from the closely related A and B virions.Infection with influenza A or B often can cause a highly contagious,acute respiratory illness.

Influenza viruses have a major impact on morbidity leading to increasesin hospitalization and in visits to health care providers. High rates ofhospitalization are observed for patients over 65 years of age and alsofor children less than 5 years of age. Influenza virus is also uniqueamong respiratory viruses in being a cause of excess mortality.Furthermore, the spread of influenza virus through a population canresult in epidemics which have considerable economic impact. Forexample, high rates of mortality were observed due to influenzainfection during the influenza epidemics of 1957, 1968 and 1977. FieldsVirology, Second Edition, Volume 1, pp. 1075-1152 (1990).

There are relatively few known compounds that have significantanti-viral activity against influenza viruses. Two of these, amantadineand rimantadine are approved in the United States for the treatment ofinfluenza virus disease. Both compounds are most effective when usedprophylactically and influenza viruses develop resistance to bothcompounds rapidly. See U.S. Pat. No. 3,152,180 and 3,352,912. Othercompounds reported to have activity against influenza viruses aredisclosed in U.S. Pat. Nos. 3,483,254, 3,496,228, 3,538,160, 3,534,084and 3,592,934.

Insofar as is known, pyridazine derivatives have not been previouslyreported as being useful for the treatment of influenza infection.

SUMMARY OF THE INVENTION

In accordance with one aspect, the present invention provides compounds,including isomeric forms, of the following structure:

wherein R₁ represents a lower alkyl (C₁-C₆) substituent which may bestraight or branched; R₂ represents an aryl substituent of the formula:

V represents a substituent selected from the group consisting of COOR₃,CONR₄R₅, SO₂NR₆R₇ and

W, X, Y and Z represent the same or different substituents selected fromthe group consisting of H, alkyl, halogen, CF₃, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl COOR′ and CONR″R′″; Q and the carbon atomsto which it is attached represent a heterocyclic ring selected from thegroup consisting of

wherein the bond between positions a, b of said heterocyclic ring formsa common bond with aromatic ring (Ar); R₃ and R′ are the same ordifferent and represent H or an alkyl (C₁-C₆) substituent; R₄, R₅, R₆,R₇, R″ and R′″ are the same or different and represent H, an alkylsubstituent, an aryl substituent, an aralkyl substituent, a heterocyclicsubstituent, a heterocyclicalkyl substituent, or a carboxyalkylsubstituent, said aryl substituent and the aryl moiety of said aralkylsubstituent having the formula:

wherein Q, V, W, X, Y and Z are as previously defined, said heterocylicsubstituent or the heterocylic moiety of said heterocyclicalkylsubstituent having the formula

wherein A is selected from the group consisting of carbon, nitrogen,sulfur or oxygen, and R₈, R₉, R₁₀, R₁₁ are the same or different andrepresent H, alkyl, halogen, CF₃, alkoxy, alkylthio, OH, alkylamino,dialkylamino, COOH, CONH₂ and SO₂NH₂, and the isomers andpharmaceutically acceptable salts of said compound.

Included within the invention also are the pharmaceutically acceptablesalts of the above compounds.

According to still another aspect, the present invention providespharmaceutical compositions comprising one or more of theabove-described pyridazine derivatives in combination with apharmaceutically acceptable carrier medium.

In accordance with yet another aspect, the present invention provides amethod for treating viral influenza infections in mammalian hosts byadministering an effective amount of the compounds of the invention to apatient susceptible to influenza infection or suffering from such aninfection.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the invention can be conveniently prepared from knownstarting materials and specific embodiments of anti-influenza compoundswithin the scope of the invention are exemplified below.

In vitro studies demonstrating the usefulness of the compounds of theinvention as anti-viral agents against the influenza virus have beenperformed. Anti-viral activity was measured on the basis of inhibitionof influenza virus transcriptase, reduction in plaque formation by theinfluenza virus and reduction in cleavage of cap 1 RNA by the influenzavirus. In addition, the effect of the anti-influenza compounds on cellgrowth was measured using a tetrazolium salt (MTT) method. Finally, drugacute tolerance was measured using studies on mice. These biologicalstudies of the anti-viral activity of the compounds of the invention aredescribed in the examples that follow.

Among the particularly preferred embodiments of the invention arecompounds, including isomeric forms, having the formula:

wherein R₁ represents CH₃; R₂ represents

V represents a substituent selected from the group consisting of COOH₃,SO₂NR₄R₅ and

R₄ and R₅ are the same or different and represent H, acetyl, methyl,substituted or unsubstituted phenyl, or substituted or unsubstitutedpyridyl, said phenyl and said pyridyl substituents being selected fromthose consisting of alkyl, alkoxy, hydroxy, carboxy and halogen groups;W represents a substituent selected from the group consisting of H, CH₃or Cl; X, Y and Z represent H; and the pharmaceutically acceptable saltsof said compounds.

Also preferred are compounds, including isomeric forms, having theformula:

wherein R₁ represents CH₃; R₂ repesents

Q and the carbon atoms to which it is attached represent a heterocyclicring selected from the group consisting of

wherein the bond between positions a, b of said heterocyclic ring formsa common bond with aromatic ring (Ar); and the isomers andpharmaceutically acceptable salts of said compound.

The term “alkyl” as used herein refers to aliphatic hydrocarbon radicalsof one to six carbon atoms in length. Similarly, the term “alkyl”, orany variation thereof, used in combination form to name substituents,such as alkoxy (—O-alkyl), alkylthio (—S-alkyl), alkylamino (—NH-alkyl),alkylsulfonyl (—S(O)₂-alkyl), carboxyalkyl (-alkyl-COOH), or the like,also refers to aliphatic hydrocarbon radicals of one to six carbon atomsin length, and preferably of one to four carbon atoms in length.

Isomers of the compound of Formula I, above, that are within the scopeof the invention include, without limitation, tautomeric forms of suchcompound.

As previously noted, the compounds of Formula I, above, including theirpharmaceutically acceptable salts, exhibit antiviral activity againstinfluenza virus.

The compounds of the invention can form salts with inorganic and organicbases, including, for example, alkali metal salts, such as Na or Ksalts, alkaline earth metal salts, such as Ca or Mg salts, ammonium,substituted ammonium and other amine salts such as morpholine,piperidine or pyridine salts.

The pharmaceutically acceptable salts of the compounds of formula I areprepared following procedures which are familiar to those skilled in theart.

The antiviral pharmaceutical compositions of the present inventioncomprise one or more of the compounds of formula I above, as the activeingredient in combination with a pharmaceutically acceptable carriermedium or auxiliary agent.

The composition may be prepared in various forms for administration,including tablets, caplets, pills or dragees, or can be filled insuitable containers, such as capsules, or, in the case of suspensions,filled into bottles. As used herein, “pharmaceutically acceptablecarrier medium” includes any and all solvents, diluents, or other liquidvehicle, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W.Martin (Mack Publishing Co., Easton, Pa., 1975) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the anti-viralcompounds of the invention, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition, its use iscontemplated to be within the scope of this invention. In thepharmaceutical compositions of the invention, the active agent may bepresent in an amount of at least 0.1% and not more than 50% by weightbased on the total weight of the composition, including carrier mediumand/or auxiliary agent(s). Preferably, the proportion of active agentvaries between 0.1 to 5% by weight of the composition. Pharmaceuticalorganic or inorganic solid or liquid carrier media suitable for enteralor parenteral administration can be used to make up the composition.Gelatine, lactose, starch, magnesium, stearate, talc, vegetable andanimal fats and oils, gum, polyalkylene glycol, or other known carriersfor medicaments may all be suitable as carrier media.

The compounds of the invention may be administered using any amount andany route of administration effective for attenuating infectivity of theinfluenza virus. Thus, the expression “amount effective to attenuateinfectivity of influenza virus”, as used herein, refers to a nontoxicbut sufficient amount of the antiviral agent to provide the desiredtreatment of viral infection. The exact amount required will vary fromsubject to subject, depending on the species, age, and general conditionof the subject, the severity of the infection, the particular antiviralagent, its mode of administration, and the like. The anti-influenzacompounds of the invention are preferably formulated in dosage unit formfor ease of administration and uniformity of dosage. The expression“dosage unit form” as used herein refers to a physically discrete unitof anti-viral agent appropriate for the patient to be treated. Eachdosage should contain the quantity of active material calculated toproduce the desired therapeutic effect either as such, or in associationwith the selected pharmaceutical carrier medium. Typically, theanti-viral compounds of the invention will be administered in dosageunits containing from about 5 mg to about 500 mg of the anti-viral agentwith a range of about 0.1 mg to about 50 mg being preferred.

The compounds of the invention may be administered orally, parenterally,such as by intramuscular injection, intraperitoneal injection, aerosol,intravenous infusion or the like, depending on the severity of theinfection being treated. The compounds of the invention may beadministered orally or parenterally at dosage levels of about 0.1 mg/kgto about 50 mg/kg and preferably from about 2 mg/kg to about 25 mg/kg,of patient body weight per day, one or more times a day, to obtain thedesired therapeutic effect.

Although the pyridazine derivatives described herein can be administeredto any patient which is susceptible to influenza infection, thecompounds are intended for the treatment of mammalian hosts, andespecially humans.

The compounds of the invention will typically be administered from 1 to3 times a day so as to deliver the above-mentioned daily dosage.However, the exact regimen for administration of the compounds andcompositions described herein will necessarily be dependent on the needsof the individual patient being treated, the type of treatmentadministered and the judgment of the attending physician.

In view of the inhibitory effect on influenza virus transcriptaseproduced by the compounds of the invention, it is anticipated that thesecompounds will be useful not only for therapeutic treatment ofinfection, but for influenza viral prophylaxis, as well. The above-noteddosages will be essentially the same whether for treatment orprophylaxis of influenza infection.

The following examples are provided to describe the invention in furtherdetail. These examples, which set forth the best mode presentlycontemplated for carrying out the invention, are intended to illustrateand not to limit the invention.

Examples 1 to 10 illustrate the chemical synthesis of ten compoundswhich are considered representative embodiments of the invention. In theexamples below in which acidification was carried out, the intermediatesor the compounds of the invention were acidified to pH 3.0. Theexpression “concentrated hydrochloric acid”, as used in the examples,refers to 3 N HCl. Also in the examples below, “excess triethylamine”means 0.5 ml triethylamine when less than one gram of compound is beingextracted or purified, and “excess triethylamine” means 1 mltriethylamine when 1-1.5 grams of compound is being extracted orpurified, based on the calculated theoretical yield.

EXAMPLE 1 Preparation of3-methyl-4-(6-methyl-3,4,5-trioxo-2H,3H,4H,5H-pyridazinyl)benzoic acid

(a) Preparation of3-methyl-4-[N′-(2-ethoxycarbonyl-1-acetyl-ethylidene)hydrazino]benzoicacid

A mixture of 3 g. (19.8 mmol) of 4-amino-3-methylbenzoic acid in 50 ml.of water and 50 ml. of ethanol and 3.56 ml. of concentrated hydrochloricacid was cooled in an ice bath and then 1.5 g. of NaNO₂ (21.8 mmol.) in10 ml. of water was added portionwise. The mixture was allowed to cometo room temperature and then added to a solution of 4.06 g. (21.8 mmol.)of ethyl 3-acetyl-4-oxopentanoate and 8 ml. of pyridine in 25 ml. ofethanol. The reaction mixture was left for 24 hours at room temperaturewith stirring. The mixture was acidified with concentrated hydrochloricacid and diluted with 20 ml. of water. The resulting solid was collectedand washed with water and pentane to yield 5.2 g.

(b) Preparation of 3-methyl-4-(3-acetyl-5-oxo-2-pyrazolin-1-yl)benzoicacid

To a solution of a 5 g. (34 mmoles) of3-methyl-4-[N′-(2-ethoxycarbonyl-1-acetylethylidene)hydrazino]benzoicacid in 25 ml. of ethanol and 25 ml. of water was added with stirring34.3 ml. of a 1 M sodium carbonate solution. The mixture was stirred atroom temperature for 24 hours. The resulting mixture was acidified to pH3 with 6 M hydrochloric acid and the resulting solid was collected byfiltration, washed with water and dried. The3-methyl-4-(3-acetyl-5-oxo-2-pyrazolin-1-yl)benzoic acid has meltingpoint of >250° C.

(c) Preparation of 2-(4-carboxy-2-methylphenyl)2,3,4,5-tetrahydro-6-methyl-pyridazine-3,4,5-trione

A solution of 3 g (11 mmoles) of3-methyl-4-(3-acetyl-5-oxo-2-pyrazodin-1-yl)benzoic acid and 8.9 g. (55mmoles) of FeCl₃ in 100 ml of acetic acid was heated to reflux for 12hours. The solution was concentrated to dryness under vacuum. Theresidual solid was suspended in water and then triethylamine was addeduntil a solution resulted. The excess triethylamine was removed in vacuoand to the solution was added 4.3 g. (55 mmoles) of sodium sulfide andthe mixture stirred at room temperature for 5 hours. The suspended solidwas removed by filtration through celite and the filtrate was acidifiedwith 6 N hydrochloric acid. The resulting mixture was centrifuged andthe supernatant liquid was discarded. The solid was resuspended and themixture recentrifuged. Process was repeated a third time and finally thesuspended solid filtered through a sintered glass funnel and washedrepeatedly with water and dried to give 2.8 g. of dark solid.

EXAMPLE 2 Preparation of the Sodium Salt of3-methyl-4-(6-methyl-3,4,5,-trioxo-2H,3H,4H,5H-pyridazinyl)benzoic acid

The sodium salt of3-methyl-4-(6-methyl-3,4,5,-trioxo-2H,3H,4H,5H-pyridazinyl)benzoic acidwas prepared as follows. Six hundred mg. of3-methyl-4-(6-methyl-3,4,5,-trioxo-2H,3H,4H,5H-pyridazinyl)benzoic acidwas dissolved in 10 ml. of water/methanol, and to the solution was addedexcess triethylamine. The excess triethylamine was removed in vacuo andthe solution passed through a 12 cm×1 cm column packed with BioRad AG 50W-X8 resin, sodium form, and eluted with 3/1 water/methanol. The eluentwas concentrated to dryness and the solid dried to give 501 mg. of darksolid.

EXAMPLE 3 Preparation of2-chloro-4-(6-methyl-3,4,5-trioxo-2H,3H,4H,5H-pyridazinyl)benzoic acid

(a) Preparation of2-chloro-4-[N′-(2-ethoxylcarbonyl-1-acetylethylidene)hydrazino]benzoicacid

To a suspension of 1 gm. (5.8 mmoles) of 3-amino-4-chlorobenzoic acid in20 ml. of ethanol was added 5 ml. of water and 1 ml. of 12 Nhydrochloric acid. The resultant solution was cooled in an ice bath andto the cooled solution was added in small portions 442 mg. (6.4 mmoles)of sodium nitrite in 3 ml. of water. The mixture was allowed to warm toroom temperature and after 30 minutes was added to a suspension of 1.19gm. (6.4 mmole) of ethyl 3-acetyl-4-oxopentanoate, 1.8 gm. of sodiumacetate, 20 ml. of ethanol and 5 ml. of water. The reaction mixtureturned dark orange. After stirring for one hour, the mixture wasacidified with 3 N hydrochloric acid and the resultant solids collectedby filtration. After drying the material, 2.53 g. was obtained.

(b) Preparation of 2-chloro-4-(3-acetyl-5-oxo-2-pyrazolin-1-yl)benzoicacid

To a suspension of 1.9 gm. (5.8 mmoles) of2-chloro-4-[N′-(2-ethoxycarbonyl-1-acetylethylidene)hydrazino]benzoicacid in 20 ml of ethanol was added at room temperature 6 ml. of aqueous1 M sodium carbonate. The mixture was left at roomtemperaturetemperature overnight. The resulting mixture was acidifed to pH 3 with 6M hydrochloric acid and the resulting solid collected by filtration anddried. The 2-chloro-4-(3-acetyl-5-oxo-2-pyrazolin-1-yl)benzoic acid hada melting point of >250° C.

(c) Preparation of 2-(4-carboxyl-3-chlorophenyl)2,3,4,5-tetrahydro-6-methyl-pyridazine-3,4,5-trione

A solution of 281 mg. (1 mmol) of2-chloro-4-(3-acetyl-5-oxo-2-pyrazolin-1-lyl)benzoic acid and 800 mg. (5mmoles) of ferric chloride were heated for 12 hours at 100° C. Thesolvent was removed in vacuo, and the residue was suspended in water andthe solid filtered and washed repeatedly with water. The solid wassuspended in water, the suspension made basic to pH 9 with 5% sodiumhydroxide followed by 1.2 gm. of sodium sulfide and the mixture stirredfor 12 hours. The mixture was filtered through filtercell and thefiltrate acidified with 6 N hydrochloric acid. The mixture wascentrifuged and the water decanted from the mixture. The solid wasslurried with water and centrifuged a second time. The process wasrepeated a third time and the dark solid dried to give 88 mg. ofmaterial which had a melting point of >300° C.

EXAMPLE 4 Preparation of4-(6-methyl-3,4,5-trioxo-2H,3H,4H,5H-pyridazinyl)benzene sulfonamide

(a) Preparation of 4-[N′-(2-ethoxycarbonyl-1-acetylethylidene)hydrazino]benzene sulfonamide

To a suspension of 10 g. (58.1 mmoles) of 4-aminobenzenesulfonamide in50 ml of 1:1 ethanol/water was added 7.3 ml. of concentratedhydrochloric acid. To the cooled mixture was added in portions 4.41. g(63.9 mmoles) of sodium nitrite in 5 ml. of water. The mixture wasallowed to come to room temperature and after 15 minutes was poured intoa solution of 11.9 g. (63.9 mmoles) of ethyl-3-acetyl-4-oxopentanoate in12.2 ml. of pyridine, in 25 ml. of ethanol. An orange solid began toseparate which was collected after 30 minutes by filtration. Afterdrying, 24.3 g. of material was obtained.

(b) Preparation of 4-(3-acetyl-5-oxo-2-pyrazolin-1-yl)benzenesulfonamide

To a solution of 24 g. (58.1 mmoles) of the hydrazone prepared inExample 4(a) above in 100 ml. of ethanol was added 60 ml. of 1 M sodiumcarbonate solution. The mixture was stirred at room temperature for 24hours and then acidified with 6 M hydrochloric acid. The resulting solidwas collected by filtration, washed with ether and dried. The amount ofproduct obtained was 3.4 g.

(c) Preparation of4-(6-methyl-3,4,5-trioxo-2H,3H,4H,5H-pyridazinyl)benzene sulfonamide

To a suspension of 1.5 g. (4.09 mmole) of4-(3-acetyl-5-oxo-2-pyrazolin-1-yl)benzene sulfonamide in 5 ml. ofacetic acid was added 1.94 g. (12 mmoles) of FeCl₃ and the mixture washeated to 90° C. for 12 hours. After cooling, the solids were collectedby filtration and washed with water and dried. The material was thendissolved in 10 ml. of water and triethylamine and 2 g. of sodiumsulfide added. After 2 hours, the mixture was acidified with 6 Nhydrochloric acid and the solid collected by centrifugation, there wasobtained 1.1 g. of material.

EXAMPLE 5 Preparation of the Sodium Salt of4-(6-methyl-3,4,5-trioxo-2H,3H,4H,5H-pyridazinyl)benzoic sulfonamide

The sodium salt of4-(6-methyl-3,4,5-trioxo-2H,3H,4H,5H-pyridazinyl)benzoic sulfonamide wasprepared by dissolving 600 mg. of the sulfonamide in methanol and addingexcess triethylamine. The excess triethylamine and methanol were removedin vacuo and the resulting solid dissolved in a mixture of 20% methanoland 80% deionized water. The solution was passed through a Bio-Rad Ag50W-XS ion exchange resin (Na form). The eluent was collected andevaporated to dryness to yield 427 mg. of material.

EXAMPLE 6 Preparation of 2-(4-tetrazolylphenyl)2,3,4,5-tetrahydro-6-methyl-pyridazine-3,4,5-trione

(a) Preparation of Ethyl3-(4-(2-tetrazolyl)-phenylhydrazino)-4-oxopentanoate

A solution of 1.06 gm. (6.58 mmoles) of 2-(4-aminophenyl)tetrazole in 20ml. of ethanol and 1.18 ml. of concentrated hydrochloric acid and 10 ml.of water was cooled in an ice bath and treated dropwise with a solutionof 500 mg. of sodium nitrite in 10 ml. of water. After the addition ofan additional 10 ml. of water, the mixture was stirred for 25 minutes atroom temperature. The mixture was then added to a solution of 1.35 gm.(7.2 mmoles) of ethyl 3-acetyl-4-oxopentanoate and 2.66 ml. of pyridinein 15 ml. of ethanol. A solid began to separate. After 1 hour, 10 ml. of1 M hydrochloric acid was added to adjust the pH to 2-3. An additional50 ml. of water was added and the solid was collected and washedthoroughly with water and dried. 1.74 g. was obtained.

(b) Preparation of3-acecyl-l-(4-tetrazolylphenyl)-4,4-dihydro-1H-pyrazol-5-one

To a solution of 1.5 gm. (4.7 mmoles of ethyl3-(4-(2-tetrazolyl)-phenylhydrazino)-4-oxopentanoate in 20 ml. ofethanol was added 5.22 mls. of a 1 M aqueous sodium carbonate solutionand the solution stirred for 12 hours at room temperature. The reactionmixture was treated with 15 ml. of 1 M hydrochloric acid followed by 30ml. of water. The resultant precipitate was collected by filtration,washed with water and hexane and dried. 1.35 g. of the intermediateproduct was obtained.

(c) Preparation of 2-(4-tetrazolylphenyl)2,3,4,5-tetrahydro-6-methyl-pyridazine-3,4,5-trione

A mixture of 350 mg. (1.3 mmoles) of the intermediate product preparedin Example 6(b), above, and 1.05 g. (6.5 mmoles) of FeCl₃ was heated to85-90° C. for 12 hours. The mixture was concentrated to dryness and theresidue suspended in water and the solid collected by filtration. Thefilter cake was dissolved in a mixture of 50% water and 50% methanolcontaining 1 ml. of triethylamine. The solution was concentrated todryness, the solid redissolved in methanol and the solution concentratedto dryness to remove excess triethylamine; the residue was dissolved in20 ml. of deionized water and to the solution was added 1.4 g. of sodiumsulfide.9H₂O. The mixture was stirred for 45 minutes and filteredthrough celite. The celite was rinsed with water. The filtrate wasacidified with 15 ml. of 1 M hydrochloric and the mixture maintainedunder vacuum to remove the evolving hydrogen sulfide gas. The mixturewas then centrifuged, the supernatant discarded and the solidresuspended in water and recentrifuged. The process was repeated threetimes and the solid finally dried. 128 mg. of dark brown solid wasobtained.

EXAMPLE 7 Preparation of the Sodium Salt of 2-(4-tetrazolylphenyl)2,3,4,5-tetrahydro-6-methyl-pyridazine-3,4,5-trione

The sodium salt of 2-(4-tetrazolyl phenyl)2,3,4,5-tetrahydro-6-methyl-pyridazine-3,4,5-trione was prepared in thefollowing manner. A 600 mg. sample of the product of Example 6 wasdissolved in a mixture of methanol/water (1:3) and triethylamine andthen the solution was concentrated to dryness to remove excesstriethylamine. The resultant solid was dissolved in a mixture ofwater/methanol (75/25), and the solution passed through a 12 cm×1 cmcolumn packed with BioRad AG 50 W-X8 resin, sodium form, and eluted with75/25 water/methanol. The eluent was concentrated to dryness and thesolid dried.

Other pyridazine derivatives and their salts as exemplified in Examples6 and 7, above can be prepared using the same general methods describedtherein.

EXAMPLE 8 Preparation of5-indazolyl-2,3,4,5-tetrahydro-6-methyl-pyridazine-3,4,5-trione

(a) Preparation of Ethyl 3-(5-indazolylhydrazino)-4-oxopentanoate

A solution of 5-aminoindazole in 50 ml. of ethanol, 100 ml. of water and8 ml. of 12 M hydrochloric acid was cooled to 0° C. and a previouslycooled solution of 3.41 g. (49.5 mmoles) of sodium nitrite in 10 ml. ofwater was added dropwise. After 30 minutes, the dark red mixture wasadded to the solution of 9.2 g, (49.5 mmoles) of ethyl3-acetyl-4-oxopentanoate in 20 ml. of ethanol and 14.2 ml. of pyridine.The resulting mixture was stirred at 0° C. for 30 minutes and then atroom temperature for an additional 30 minutes and finally the solidcollected by filtration to give 11.2 g. of solid.

(b) Preparation of 3-acetyl-1-(5-indazolyl)-4,4-dihydro-1H-pyrazol-5-one

A solution of 10.37 g. (36 mmoles) of ethyl3-(5-indazolylhydrazino)-4-oxopentanoate in 40 ml. of a 1 M solution ofsodium carbonate, 40 ml. of water and 40 ml. of ethanol was stirred atroom temperature for 12 hours. The solution was diluted with 200 ml. ofwater and acidified to pH 3 with 1 N hydrochloric acid. The brown solidwhich separated was collected to give 7.16 g. of product.

(c) Preparation of5-indazolyl-2,3,4,5-tetrahydro-6-methyl-pyridazine-3,4,5-trione

A solution of 242 mg (1 mmole) of3-acetyl-1-(5-indazolyl)-4,4-dihydro-1H-pyrazol-5-one and 810 mg. (5mmoles) of FeCl₃ in 10 ml. of acetic acid was heated to 90° C. for 12hours. The acetic acid was removed under vacuum and 15 ml. of water wasadded to the residue. The solid was collected by filtration and thendissolved in 100 ml. of 1:1 methanol/water. Triethylamine was addeduntil the solution was basic and the solution concentrated under vacuumto remove excess triethylamine. The solution was diluted to 30 ml. andthe 1 g. of sodium sulfide added. After stirring for 2 hours the solidwas removed by filtration through celite. The filtrate was acidifiedwith 1 N hydrochloric acid to pH 2 and the mixture centrifuged. Thesupernatant liquid was decanted from the mixture and the remaining solidwas slurried with water and centrifuged a second time and the solidcollected and dried to give 140 mg. of product.

This is a specific representative example of a compound of Formula I,above, in which Q and the carbon atoms to which it is attached representa heterocyclic ring (pyrazole), with the bond between positions a, b ofthe heterocyclic ring forming a common bond with aromatic ring (Ar).

EXAMPLE 9 Preparation of5-benzotriazolyl-2,3,4,5-tetrahydro-6-methyl-pyridazine-3,4,5-trione

(a) Preparation of Ethyl 3-(5-benzotriazolylhydrazino)-4-oxopentanoate

To a solution of 1.0 g. (7.45 mmoles) of 5-aminobenzotriazole in 10 ml.of ethanol, was added 10 ml. of water and 45 ml. of concentratedsulfuric acid. The solution was cooled to 0° C. and a solution of 560mg. (8.2 mmoles) of sodium nitrite in 3 ml. of water was added dropwise.After 90 minutes at this temperature, the solution was added to asolution of 1.53 g. (8.2 mmoles) of 3-acetyl-4-oxopentanoate, 2.05 g.(22.3 mmoles) and sodium acetate in 10 ml. of ethanol and 20 ml. ofwater. A solid began to separate which was collected after 30 minutes togive 1.73 g. of product.

(b) Preparation of3-acetyl-1-(5-benzotriazolyl)-4,4-dihydro-1H-pyrazol-5-one

To a suspension of 1.73 g. (5.98 mmoles) of ethyl3-(5-benzotriazolylhydrazino)-4-oxopentanoate in 20 ml. of etnanol wasadded 9 ml. of 1 M sodium carbonate. The solution was stirred for 12hours and after acidification with 6 N hydrochloric acid, the resultingsolid was collected and dried to give 820 mg. of product.

(c) Preparation of5-benzo-triazolyl-2,3,4,5-tetrahydro-6-methyl-pyridazine-3,4,5-trione

To a solution of 485 mg (1.99 mmole) of3-acetyl-1-(5-benzotriazolyl)-4,4-dihydro-1H-pyrazol-5-one in 5 ml. ofacetic acid was added 1.61 g. (9.95 mmoles) of FeCl₃. The solution washeated to 90° C. for 12 hours. The solution was diluted with 50 ml. ofwater and the solid which separated was washed with water and dried. 170mg. of dark solid was obtained.

This is another specific representative example of a compound of FormulaI, above, in which Q and the carbon atoms to which it is attachedrepresent a heterocyclic ring (triazole), with the bond betweenpositions a,b of the heterocyclic ring forming a common bond witharomatic ring (Ar.) Examples 10-12 illustrate the efficacy of compoundsof the invention in inhibiting viral transcriptase activity, ininhibiting plaque formation by the influenza virus and in inhibitingcleavage of cap 1 RNA by the influenza virus.

EXAMPLE 10

Assay for Influenza A/WSN Virus Transcription

The assay for influenza A/WSN virus transcription was performed withdetergent-treated purified influenza virions and 2′-O-methylated alfalfamosaic virus RNA4 (AlMV RNA4) according to the following procedure.Duplicate reactions (50 μl in 96 well polypropylene U-bottom plates)contained 50 mM Hepes, pH 8, 50 mM potassium acetate, 5 mMdithiothreitol (DTT) , 5 mM magnesium chloride, 1% Triton N-101, 35 μMATP, 0.3 μM CTP,. 0.1 μM GTP, 1 μM UTP, 2 μCi 35S-UTP (Amersham SJ1303),0.75 μg (15 μg/ml) purified virions, and 5 ng (0.4 nM) cap 1 AlMV RNA4.Test compounds were solubilized with 100% dimethylsulfoxide (DMSO) andwere present in the reactions at 1% DMSO. The reference standardinhibitor, poly (A, G), was present at concentrations of 10, 3, 1, 0.3,and 0.1 μg/ml. Incubation was for 45 minutes at 31° C. Reactions werestopped by the addition of 150 μl of ice-cold 7% trichloracetic acid(TCA)+2% sodium pyrophosphate containing 50 μg/ml yeast tRNA. The TCAprecipitates were filtered onto Millipore HATF plates pre-wetted with200 μl of 7% TCA+2% sodium pyrophosphate without yeast tRNA. Plates werewashed four times with 5% TCA+2% sodium pyrophosphate and filters weredried and coated with Wallac Meltilex A. Scintillant-backed filters werepunched onto Fascol marking film, sealed and quantitated using a Wallac1450 MicroBeta scintillation counter. Alternatively, a MolecularDynamics Storm System was used; in this case, the filters were notbacked with solid scintillant but were quantitated directly.

The results given in Table 1 were measured as the IC₅₀ or theconcentration of drug compound required to achieve a 50 inhibition ofinfluenza A/WSN virus transcriptase activity.

TABLE 1 Example Number IC₅₀ (μM) 1 0.1 4 1 6 0.2

The low concentrations of drug compounds required to achieve 50%inhibition of the viral transcriptase activity indicate that the drugcompounds of the invention are effective at inhibiting the influenzaA/WSN virus transcript-ion process.

EXAMPLE 11 Assay for Antiviral Activity Against Influenza A/WSN,A/Victoria and B/Lee Viruses

Compounds were evaluated for antiviral activity against influenza A/WSN,A/Victoria and B/Lee viruses by plaque reduction in Madin Darby caninekidney (MDCK) cells. Duplicate monolayers of MDCK cells in 6 well plateswere washed free of protein-containing media, infected with 50-100plaque-forming units of virus (0.4 ml. volume), and incubated at 37° C.for 60 minutes. After aspiration of the virus inoculum, a 0.6% agaroseoverlay (3 ml.) containing Eagle minimal essential media, trypsin (8μg/ml.), and the appropriate drug dilution (final concentration of 1%DMSO) was added to the cell monolayer. Plates were incubated at 37° C.in a humidified atmosphere of 5% CO₂ in air. After 48 hours, monolayerswere fixed with glutaraldehyde, stained with 0.1% crystal violet and theplaques were counted. The percentage of plaque inhibition relative tothe infected control (no drug) plates were calculated for each drugconcentration and the 50% inhibitory concentration (IC₅₀) wasdetermined.

The results given in Table 2 were measured as the IC₅₀ or theconcentration of compound required to achieve a 50% inhibition ofinfluenza virus plaque formation.

TABLE 2 IC₅₀ (μM) Example Number A/WSN A/Victoria B/Lee 1 50 90 >200 2100 >200 3 16 50 175 4 8 8 35 5 8 11 30 6 1 2 40 7 2 115 8 50 9 20

The plaque reduction results given in Table 2 illustrate that thecompounds of the invention exhibit antiviral activity against theinfluenza virus by inhibiting plaque formation by the influenza A/WSN,A/Victoria and B/Lee viruses.

EXAMPLE 12 Assay for Cleavage of cap 1 AIMV RNA4 by Influenza Virus

(a) Preparation of cap I RNAs containing 32P in the cap

To prepare ³²p-labeled cap 1 AIMV RNA4, the terminal m⁷G of AIMV RNA4was first removed by β-elimination (H. Fraenkel-Conrat and A.Steinschneider, Methods in Enzymology 12B, 243-246 (1967); S. J. Plotch,M. Bouloy and R. M. Drug, Proc. Natl. Acad. Sci. USA, 76, 1618-1622(1979)). Two μg of β-eliminated RNA was then incubated for 1 hour at 37°C. in a 50 μl. reaction containing 25 mM Hepes, pH 7.5, 1 mM DTT, 20units of guanylyltransferase enzyme (GIBCO/BRL #8024SA), 1 mM magnesiumchloride, 4 μCi of ³H-S-adenosylmethionine (Amersham TRK.614), and 100μCi of ³²P-GTP (Amersham PB 10201). The RNA was phenol andchloroform-extracted, separated from unincorporated radionucleotidesusing a G-50 spun column, and ethanol-precipitated prior to being addedto a cleavage reaction.

(b) Cleavage Assay

The cleavage reaction conditions were identical to the transcriptionreaction conditions except that no nucleotides were present and³²p-labeled cap 1 AIMV RNA4 was used. Cleavage reaction products werephenol and chloroform-extracted, ethanol precipitated, and resolved byelectrophoresis on 20% acrylamide-6 M urea gels. The reaction productswere quantitated using a Molecular Dynamics Storm 840 imaging system.

The results given in Table 3 were measured as the ICSO or theconcentration of compound required to achieve a 50% inhibition ofinfluenza virus cleavage of cap 1 RNA.

TABLE 3 Example Number IC₅₀ (μM) 1 0.2 4 2

The low concentrations of compounds required to achieve 50% inhibitionof the viral transcriptase activity indicate that the compounds of theinvention are effective at inhibiting cleavage of cap 1 RNA by theinfluenza virus.

Example 13 shows the effect on cell growth produced by theanti-influenza compounds of the invention.

EXAMPLE 13

Cell Growth Assay

Effects of the pyridazine derivatives of the invention on cell growthwere determined in MDCK cells in 96 well plates by a tetrazolium-basedcolorimetric method (R. Pauwels et al., J. Virol. Methods, 20, 309-321(1988)). This assay detects the in situ reduction of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) byviable cells. Approximately 1×10⁴ cells were seeded per well andincubated with drug-containing growth media for 2-3 days (3-4 celldoublings). The drug concentration resulting in a reduction of opticaldensity by 50% was determined.

The results given in Table 4 were measured as the IC₅₀ or theconcentration of compound required to achieve a 50% reduction of opticaldensity.

TABLE 4 Example Number IC₅₀ (μM) 1 >200 4 >100

These results indicate that relatively high concentrations of theanti-viral compounds are required to achieve a 50% reduction of opticaldensity which is a measure of cell growth or viability. Theconcentrations at which anti-influenza activity have been observed aremuch lower than the concentrations at which cell viability was affected.

Example 14 shows the tolerance of the drug compounds of the invention inanimal studies using mice.

EXAMPLE 14 Acute Tolerance Assay

Compounds of the invention were administered to mice and the mice werethen monitored for tolerance of the drug. The mice were monitored foradverse effects hourly during the 6 hours post-administration, and twicedaily thereafter for 2 weeks. Euthanasia was administered to mordibundand distressed animals.

The mice (5/group; Swiss Webster female, 8-9 week old, 25-30 g) receiveda single administration of compounds of the invention by either the oralgavage (0.5 mL) or tail vein injection (0.2 mL) as shown in Table 5below.

TABLE 5 Acute Tolerance Compound/ Route of Volume Group Admin- DoseAdministered/ (5 mice) istration (mg/kg) Animal Cmpd. of Ex. 2 1 Oral  00.5 mL saline 2 Gavage  21 (0.6 mg/28 g mouse) 0.5 mL 1.3 mg/mL 3  71(2.0 mg/28 g mouse) 0.5 mL 4.0 mg/mL 4 214 (6.0 mg/28 g mouse) 0.5 mL 13mg/mL 5 710 (20.0 mg/28 g mouse) 0.5 mL 40 mg/mL 6 IV Injection  0 0.2mL saline 7 (tail vein)  2 (0.06 mg/28 g mouse) 0.2 mL 0.3 mg/mL 8  7(0.2 mg/28 g mouse) 0.2 mL 1 mg/mL 9  21 (0.6 mg/28 g mouse) 0.2 mL 3mg/mL 10  71 (2.0 mg/28 g mouse) 0.2 mL 10 mg/mL Cmpd. of Ex. 5 11 Oral 0 0.5 mL saline 12 Gavage  21 (0.6 mg/28 g mouse) 0.5 mL 1.3 mg/mL 13 71 (2.0 mg/28 g mouse) 0.5 mL 4.0 mg/mL 14 214 (6.0 mg/28 g mouse) 0.5mL 13 mg/mL 15 710 (20.0 mg/28 g mouse) 0.5 mL 40 mg/mL 16 IV Injection 0 0.2 mL saline 17 (tail vein)  2 (0.06 mg/28 g mouse) 0.2 mL 0.3 mg/mL18  7 (0.2 mg/28 g mouse) 0.2 mL 1 mg/mL 19  21 (0.6 mg/28 g mouse) 0.2mL 3 mg/mL 20  71 (2.0 mg/28 g mouse) 0.2 mL 10 mg/mL

With the exception of one mouse in group 20 which died, due to causesunrelated to administration of the compound itself, 2 hours afteradministration of the compound of the invention, all the other micesurvived at least 16 days after administration of the compounds of theinvention. These results indicate that mice have a high tolerance forthe compounds of the invention.

Other compounds of the present invention that have been found to exhibitsignificant potency against influenza include (substituents given withreference to Formula I, above):4-(6-methyl-3,4,5-trioxo-2H,3H,4H,5H-pyridazinyl)benzenecarboxamide(R₁=methyl; R₂=4-amidophenyl);2-methyl-5-(6-methyl-3,4,5-trioxo-2H,3H,4H,5H-pyridazinyl)benzenesulfonamide(R₁=methyl; R₂=3-sulfonamido-4-methylphenyl);N-methyl-4-chloro-3-(6-methyl-3,4,5-trioxo-2H,3H,4H,5H-pyridazinyl)benzenesulfonamide(R₁=methyl; R₂=3-N-methylsulfonamide-6-chlorophenyl);N-methyl-4-(6-methyl-3,4,5-trioxo-2H,3H,4H,5H-pyridazinyl)benzenesulfonamide(R₁=methyl; R₂=4-N-methylphenylsulfonamido);N-phenyl-4-(6-methyl-3,4,5-trioxo-2H,3H,4H,5H-pyridazinyl)benzenesulfonamide(R₁=methyl; R₂=4-N-phenylsulfonamidophenyl);N-acetyl-4-(6-methyl-3,4,5-trioxo-2H,3H,4H,5H-pyridazinyl)benzenesulfonamide(R₁=methyl; R₂=N-acetyl sulfonamidophenyl);N-(3-pyridyl)-4-(6-methyl-3,4,5-trioxo-2H,3H,4H,5H-pyridazinyl)benzenesulfonamide(R₁=methyl; R₂=4-N-(3-pyridyl)sulfonamidophenyl); and6-(6-methyl-3,4,5-trioxo-2H,3H,4H,5H-pyridazinyl)-1,1-dioxo-1,2-dihydro-1λ⁶-benz<d>isothiazol-3-one(R₁=methyl; R₂=1,1-dioxo-1,2-dihydro-1λ⁶-benz<d>isothiazol-3-one).

Although the present invention has been described and exemplified interms of certain preferred embodiments, other embodiments will beapparent to those skilled in the art. The invention is, therefore, notlimited to the particular embodiments described and exemplified, but iscapable of modification or variation without departing from the spiritof the invention, the full scope of which is delineated by the appendedclaims.

What is claimed is:
 1. A compound produced by the process comprising theprocess step of reacting the starting compound of the formula:

with FeCl₃ in the presence of acetic acid, wherein the reaction mixtureis heated and the temperature of the reaction mixture is about 85° C. toabout reflux temperature, and wherein R₁ in the above formula representsa lower alkyl (C₁-C₆) substituent which may be straight or branched andR₂ in the above formula represents an aryl substituent of the formula:

V represents a substituent selected from the group consisting of COOR₃,CONR₄R₅, SO₂NR₆R₇ and

W, X, Y and Z represent the same or different substituents selected fromthe group consisting of H, alkyl, halogen, CF3, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl COOR′ and CONR″R′″; Q and the carbon atomsto which it is attached represent a heterocyclic ring selected from thegroup consisting of

wherein the bond between positions a, b of said heterocyclic ring formsa common bond with aromatic ring (Ar); R₃ and R′ are the same ordifferent and represent H or an alkyl (C₁-C₆) substituent; R₄, R₅, R₆,R₇, R″ and R′″ are the same or different and represent H, an alkylsubstituent, an aryl substituent, an aralkyl substituent, a heterocyclicsubstituent, a heterocyclicalkyl substituent, or a carboxyalkylsubstituent, said aryl substituent and the aryl moiety of said aralkylsubstituent having the formula:

wherein Q, V, W, X, Y and Z are as previously defined, said heterocyclicsubstituent or the heterocyclic moiety of said heterocyclicalkylsubstituent having the formula

wherein A is selected from the group consisting of carbon, nitrogen,sulfur or oxygen, and R₈, R₉, R₁₀, R₁₁ are the same or different andrepresent H, alkyl, halogen, CF₃, alkoxy, alkylthio, OH, alkylamino,dialkylamino, COOH, CONH₂ and SO₂NH₂, and the isomers andpharmaceutically acceptable salts of said compound.
 2. The compoundaccording to claim 1, wherein said reaction mixture is heated for about12 hours.
 3. The compound according to claim 1, wherein the reactionmixture is refluxed for 12 hours.
 4. The compound according to claim 1,wherein the temperature is from about 85° C. to about 100° C.
 5. Thecompound according to claim 1, wherein said temperature is from about90° C. to about 100° C.
 6. The compound according to claim 1, whereinsaid starting compound is3-methyl-4-(3-acetyl-5-oxo-2-pyrazolin-1-yl)benzoic acid.
 7. Thecompound according to claim 1, wherein said starting compound is2-chloro-4-(3-acetyl-5-oxo-2-pyazolin-1-yl)benzoic acid.
 8. The compoundaccording to claim 1, wherein said starting compound is4-(3-acetyl-5-oxo-2-pyrazdin-1-yl)benzene sulfonamide.
 9. The compoundaccording to claim 1, wherein said starting compound is3-acetyl-1-(4-trazolyl phenyl)-4,4-dihydro-1H-pyrazol-5-one.
 10. Thecompound according to claim 1, wherein said starting compound is3-acetyl-1-(5-indazolyl phenyl)4,4-dihydro-1H-pyrazol-5-one.
 11. Thecompound according to claim 1, wherein said starting compound is3acetyl-1-(5-benzotriazolyl)-4,4-dihydro-1H-pyrazol-5-one.